MicroRNAs as Modulators of Tumor Metabolism, Microenvironment, and Immune Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. Molecular heterogeneity and absence of biomarkers helping patient allocation to the best therapeutic option contribute to poor prognosis in advanced stages. MicroRNAs' (miRNAs) deregulated expression contributes to tumor development and progression and influences drug resistance in HCC. Accordingly, miRNAs have been extensively investigated as both biomarkers and therapeutic targets. The diagnostic and prognostic roles of circulating miRNAs have been ascertained, though with some inconsistencies across studies. From a therapeutic perspective, miRNA-based approaches demonstrated safety profiles and antitumor efficacy in HCC animal models. Nevertheless, caution should be used when transferring preclinical findings to the clinic, due to possible molecular inconsistency between animal models and the heterogeneous patterns of human diseases. Awealth of information is offered by preclinical studies exploring the mechanisms driving miRNAs' aberrant expression, the molecular cascades triggered by miRNAs and the corresponding phenotypic changes. Ex-vivo analyses confirmed these results, further shedding light on the intricacy of the human disease often overcoming pre-clinical models. This complexity seems to be ascribed to the intrinsic heterogeneity of HCC, to different risk factors driving its development, as well as to changes across stages and previous treatments. Preliminary findings suggest that miRNAs associated with specific risk factors might be more informative in defined patients' subgroups. The first issue to be considered when trying to envisage a possible translational perspective is the molecular context that often drives different miRNA functions, as clearly evidenced by "dual" miRNAs. Concerning the possible roles of miRNAs as biomarkers and therapeutic targets, we will focus on miRNAs' involvement in metabolic pathways and in the modulation of tumor microenvironment, to support their exploitation in defined contexts

Animal models of hepatocellular carcinoma prevention

Hepatocellular carcinoma (HCC) is a deadly disease and therapeutic efficacy in advanced HCC is limited. Since progression of chronic liver disease to HCC involves a long latency period of a few decades, a significant window of therapeutic opportunities exists for prevention of HCC and improve patient prognosis. Nonetheless, there has been no clinical advancement in instituting HCC chemopreventive strategies. Some of the major challenges are heterogenous genetic aberrations of HCC, significant modulation of tumor microenvironment and incomplete understanding of HCC tumorigenesis. To this end, animal models of HCC are valuable tools to evaluate biology of tumor initiation and progression with specific insight into molecular and genetic mechanisms involved. In this review, we describe various animal models of HCC that facilitate effective ways to study therapeutic prevention strategies that have translational potential to be evaluated in a clinical context © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell popula-tions displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas

Circulating CD8 lymphocytes predict response to atezolizumab–bevacizumab in hepatocellular carcinoma

Due to the lack of biomarkers predictive of response to atezolizumab-bevacizumab, the standard of care for advanced HCC, we analyzed baseline and early on-treatment variation of peripheral lymphocyte populations of 37 prospective patients treated by atezolizumab-bevacizumab and in 15 prospective patients treated by sorafenib or lenvatinib (TKIs). RNAseq analysis followed by RT-PCR validation on patients-derived PBMC was also performed. At first imaging, re-evaluation 13 patients receiving atezolizumab-bevacizumab, showed an objective response, 17 stable disease, while 7 were nonresponders. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes were lower in responders versus nonresponders (T-test, p = 0.012 and 0.004, respectively). At 3 weeks, 28 of 30 responders displayed a rise of CD8+PD1+ lymphocytes with a positive mean fold change of 4.35 (+/- 5.6 SD), whereas 6 of 7 nonresponders displayed a negative fold change of 0.89 (+/- 0.84 SD). These changes were not observed in patients treated by TKIs. TRIM56, TRIM16, TRIM64, and Ki67 mRNAs were validated as upregulated in responders versus nonresponders after 3 weeks after treatment start, providing possible evidence of immune activation. Baseline CD8+ and CD8+PD-L1+ peripheral lymphocytes and early changes in CD8+PD1+ lymphocytes predict response to atezolizumab-bevacizumab providing noninvasive markers to complement clinical practice in the very early phases of treatment of HCC patients.PBMCs represent important elements in assessing the response to immune checkpoint inhibitors. Their analyses through flow cytometry show that baseline CD8+, CD8+PD-L1+, and an early increase in CD8+PD1+ peripheral lymphocytes after the first drug infusion, reliably predict the response to atezolizumab-bevacizumab evaluated by imaging in advanced hepatocellular carcinoma. imag

Early CTLA4 increase in CD45+ blood cells: an emerging biomarker of atezolizumab–bevacizumab resistance and worse survival in advanced hepatocarcinoma

Background: Advanced hepatocellular carcinoma (HCC) has a dismal prognosis; however, the introduction of atezolizumabebevacizumab combination has improved overall survival and novel immune checkpoint inhibitors are entering the clinics. Despite more therapeutic options being available, no biomarker guides treatment choice. Indeed, tissue-based analyses and complex analytical procedures hinder clinical translation. We explored the informativeness of a simple, non-invasive, repeatable cytofluorimetric assay on peripheral blood to predict response and survival in HCC patients treated with atezolizumabebevacizumab. Materials and methods: Twenty-five cirrhotic patients, 50 HCC patients undergoing atezolizumabebevacizumab and an independent validation cohort of 25 HCC patients were subjected to a cytofluorimetric study of peripheral white blood cells (WBCs) to assess baseline programmed death-ligand 1-positive (PD-L1þ) and cytotoxic T-lymphocyte antigen 4positive (CTLA4þ) cell percentage in the different populations and their early on-treatment variations. Immunophenotypes were evaluated against treatment response. RNA sequencing followed by RTePCR validation were used to elucidate the molecular correlates of immunophenotypic observations. Results: PD-L1þ cell percentage did not predict response either at baseline or when evaluating treatment-induced early changes. Conversely, the percentage of CTLA4þ lymphocytes at baseline showed a predictive significance (35.37 in responders versus 31.5 in non-responders, P 1⁄4 0.03). More interestingly, the early CTLA4þ changes during treatment in lymphocytes (responders 0.95 versus non-responders 1.08, P 1⁄4 0.05), monocytes (responders 0.95 versus non-responders 1.04, P 1⁄4 0.03), granulocytes (responders 0.94 versus non-responders 1.14, P 1⁄4 0.001) and, even stronger, the early CTLA4þ percentage change in the whole WBCs displayed a predictive significance in terms of time to progression (TTP) (P < 0.0001) and overall survival (OS) (P 1⁄4 0.005). The immunophenotypic findings correlated with transcriptional modulation of CTLA4 target genes and genes involved in immune response. Conclusions: A repeatable, easy, non-invasive blood test predicts response to immunotherapy in patients with HCC, both in terms of TTP and OS. CTLA4þ cell percentage increase in non-responders suggests a possible resistance mechanism which deserves attention as a druggable target. Key words: immunotherapy, CTLA4, PD-L1, biomarker

Polymorphism AGT2 (rs4762) is involved in the development of dermatologic events: Proof-of-concept in hepatocellular carcinoma patients treated with sorafenib

BACKGROUND Dermatologic adverse events (DAEs) are associated with a better outcome in patients with hepatocellular carcinoma (HCC) irrespective of the therapeutic agent received. The exact mechanisms associated with the development of DAEs are unknown although several studies point to direct toxicity of tyrosine kinase inhibitors (TKIs) to the skin or an immune-mediated reaction triggered by the oncologic treatment. As is the case in other conditions, individual genetic variants may partially explain a higher risk of DAEs. AIM To evaluate the contribution of several gene variants to the risk of developing DAEs in HCC patients treated with TKIs. METHODS We first analyzed 27 single-nucleotide polymorphisms (SNPs) from 12 genes selected as potential predictors of adverse event (AE) development in HCC patients treated with sorafenib [Barcelona Clinic Liver Cancer 1 (BCLC1) cohort]. Three additional cohorts were analyzed for AGT1 (rs699) and AGT2 (rs4762) polymorphisms-initially identified as predictors of DAEs: BCLC2 (n= 79), Northern Italy (n= 221) and Naples (n= 69) cohorts, respectively. The relation between SNPs and DAEs and death were assessed by univariate and multivariate Cox regression models, and presented with hazard ratios and their 95% confidence intervals (95%CI). RESULTS The BCLC1 cohort showed that patients with arterial hypertension (AHT) (HR = 1.61; P value = 0.007) and/or AGT SNPs had an increased risk of DAEs. Thereafter, AGT2 (rs4762) AA genotype was found to be linked to a statistically significant increased probability of DAEs (HR = 5.97; P value = 0.0201, AA vs GG) in the Northern Italy cohort by multivariate analysis adjusted for BCLC stage, ECOG-PS, diabetes and AHT. The value of this genetic marker was externally validated in the cohort combining the BCLC1, BCLC2 and Naples cohorts [HR = 3.12 (95%CI: 1.2-8.14), P value = 0.0199, AGT2 (rs4762) AA vs AG genotype and HR = 2.73 (95%CI: 1.18-6.32) P value = 0.0188, AGT2 (rs4762) AA vs GG genotype]. None of the other gene variants tested were found to be associated with the risk of DAE development. CONCLUSION DAE development in HCC patients receiving TKIs could be explained by the AGT2 (rs4762) gene variant. If validated in other anti-oncogenic treatments, it might be considered a good prognosis marker

ER Stress Negatively Modulates the Expression of the miR-199a/214 Cluster to Regulates Tumor Survival and Progression in Human Hepatocellular Cancer

Background: Recent studies have emphasized causative links between microRNAs (miRNAs) deregulation and tumor development. In hepatocellular carcinoma (HCC), more and more miRNAs were identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools. This study aimed to investigate the functional significance and regulatory mechanism of the miR-199a2/214 cluster in HCC progression. Methods and Findings: In this study, we showed that miR-214, as well as miR-199a-3p and miR-199a-5p levels were significantly reduced in the majority of examined 23 HCC tissues and HepG2 and SMMC-7721 cell lines, compared with their nontumor counterparts. To further explore the role of miR-214 in hepatocarcinogenesis, we disclosed that the ER stressinduced pro-survival factor XBP-1 is a target of miR-214 by using western blot assay and luciferase reporter assay. Reexpression of miR-214 in HCC cell lines (HepG2 and SMMC-7721) inhibited proliferation and induced apoptosis. Furthermore, ectopic expression of miR-214 dramatically suppressed the ability of HCC cells to form colonies in vitro and to develop tumors in a subcutaneous xenotransplantation model of the BALB/c athymic nude mice. Moreover, reintroduction of XBP-1s attenuated miR-214-mediated suppression of HCC cells proliferation, colony and tumor formation. To further understand the mechanism of the miR-199a/214 cluster down-expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia-induced unfolded protein response (UPR) suppresses the expression of the miR-199a/21

MicroRNA-122 Modulates the Rhythmic Expression Profile of the Circadian Deadenylase Nocturnin in Mouse Liver

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3′-untranslated region (3′-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3′-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control